Post-transcriptional mechanisms regulating gene expression provide accurate synthesis of all functional polypeptides that are critical for cell growth and development. Soluble translation factors are essential to facilitate delivery of cognate aminoacylated tRNA (aa-tRNA) to the translating ribosome. Translation elongation requires the GTP-dependent activity of the elongation factor (eEF1) complex to promote the binding of aa-tRNA to the A-site of ribosomes during protein synthesis. Eukaryotic eEF1 A of the eEF1 complex is the second most abundant protein in the cell and belongs to the G-protein superfamily that includes the oncogene Ras. Inappropriate expression of eEF1 A has been shown in many tumors, and eEF1A has been associated with a highly metastatic potential in cancer cells. To sustain sufficient translation elongation for viability, eEF1Balpha functions as the guanine nucleotide exchange factor (GEF) for eEF1A to maintain the active GTP-bound form and continuation of the elongation of the peptide chain. Current models suggest that the action of eEF1Balpha on eEF1A regeneration is the rate-limiting step of translation elongation. Furthermore, the eEF1A:GTP:eEF1Balpha intermediate complex may be responsible for interacting directly with aa-tRNA. Utilizing yeast Saccharomyces cerevisae as a model system with a set of coordinated structural, kinetic, and biochemical approaches, we will address key questions regarding the responsibility of eEFIBalpha in the maintenance of translational elongation and roles other than its GEF function. This proposed work aims to determine how eEF1Balpha affects the activity of eEF1A such as GTP binding to eEF1A, formation of the eEF1 A:GTP:aa-tRNA ternary complex, and the ternary complex recruitment to the ribosome using established fluorescent-based studies, a ribonucluease protection assay, and gel filtration chromatography, respectively. To further dissect the critical role of eEF1Balpha, the proposal work will utilize structural-based mutations of eEF1Balpha to increase our understanding of the GEF mechanism by stopped-flow kinetics and the interplay between eEF1A:eEF1Balpha complex formation and other steps of the elongation that may potentially involve the GEF eEF1Balpha. Therefore, this research proposal focuses on understanding how eEF1Balpha contributes in regulating and facilitating the translational activity of eEF1A, thereby providing us with specific insight into G-protein regulation. [unreadable] [unreadable] [unreadable]